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Analysis of DNA strand break repair using human knockout cells

使用人类敲除细胞分析 DNA 链断裂修复

基本信息

批准号：
18590063
负责人：
ADACHI Noritaka
金额：
$ 2.49万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590063/
关键词：
DNA strand break DNA repair end-ioining random integration gene targeting nonhomologous recombination 非相同組換之

项目摘要

We recently developed a system using the human cell line Nalm-6 that enables rapid production of human gene knockout cells. In this study, we used this system to genetically analyze the function of human genes that have been implicated in DNA strand break repair. Specifically, we disrupted a series of genes involving those for Rad54, FancB, Mus81, Tdp1, Ku70/80, and Lig3, which all have some roles in DNA single and/or double-strand break repair in human cells. In addition, we tried to produce as many double-knockout mutant cells as possible, particularly those with a Lig4 mutation. Using these mutants, we have successfully analyzed the cellular sensitivity to anticancer agents involving cisplatin and topoisomerase inhibitors as well as the genetic interaction between p53 and BLM and between Mus81 and FancB, We also performed gene knockdown experiments with siRNAs for DNA ligases that are potentially involved in random integration and/or gene targeting. The results suggested that suppressing nonhomologous end-joining reactions could enhance the efficiency of gene targeting. Our findings also indicated that there must be another mechanism for random integration that does not rely on the nonhomologous end-joining pathway.

我们最近开发了一种使用人类细胞系Nalm-6的系统，该系统能够快速生产人类基因敲除细胞。在这项研究中，我们使用该系统从遗传学上分析了与DNA链断裂修复有关的人类基因的功能。具体来说，我们破坏了一系列涉及Rad 54，FancB，Mus 81，Tdp 1，Ku 70/80和Lig 3的基因，这些基因在人类细胞中的DNA单链和/或双链断裂修复中都有一定的作用。此外，我们试图产生尽可能多的双敲除突变细胞，特别是那些具有Lig 4突变的细胞。使用这些突变体，我们已经成功地分析了细胞对涉及顺铂和拓扑异构酶抑制剂的抗癌剂的敏感性，以及p53和BLM之间以及Mus 81和FancB之间的遗传相互作用。我们还用siRNA对可能参与随机整合和/或基因靶向的DNA连接酶进行了基因敲减实验。结果表明，抑制非同源末端连接反应可以提高基因打靶的效率。我们的研究结果还表明，必须有另一种机制的随机整合，不依赖于非同源末端连接途径。